This proposal outlines experiments to study the roles played by the West Nile virus envelope proteins in viral fusion with the cell. We propose a large-scale genetic and functional analysis of the envelope proteins using our novel approach of genetic footprinting, a method that we have developed and applied successfully to other viruses. In contrast to most conventional methods of mutagenesis, which involve the isolation, storage and characterization of each mutant separately, genetic footprinting allows for efficient construction and parallel functional analysis of thousands of mutations. Our objective is to identify domains in these proteins that are critical for viral entry into host cells. Such detailed functional mapping will allow us to begin to gain a mechanistic understanding of viral fusion. Aim 1 consists of creating comprehensive, near-saturating libraries of mutations in the E and prM/M envelope proteins of West Nile virus, using a transposon-based mutagenesis method. Aim 2 consists of a parallel functional analysis of the library of mutations to define regions of the envelope proteins that are necessary for viral fusion. Aim 3 consists of analyzing individual mutations in envelope proteins to obtain a detailed understanding of the mechanism of viral entry. We present data to show the successful generation of a library of insertional mutations in an infectious clone of WNV that contains a fluorescent marker. We also present details of novel cell-cell fusion assay that we have set up using hepatitis C virus (another flavivirus) envelope proteins, as preliminary data. A similar fusion assay will be developed for WNV. Identification of domains essential for fusion will help in selecting regions for further analysis, and in understanding the process of viral entry. This information will be useful in generating better vaccines and in targeting the fusion process with small molecule inhibitors. Relevance to Public Health: Since its introduction to North America in 1999, West Nile virus has spread to most of the US, causing neurological damage and death in some infected individuals. The epidemic continues to change in character and is still not understood. Lack of specific treatment or a licensed vaccine make it especially important that we understand the fundamental processes by which the virus infects the host cell. Our comprehensive study of the envelope proteins will provide such information with the eventual goal of being able to block viral infection using drugs and vaccines. [unreadable] [unreadable] [unreadable]